Abstract Type-2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia, relative insulin deficiency and insulin resistance. Insulin resistant cells show defects in insulin-induced exocytosis of the glucose transporter GLUT4, affecting glucose uptake. The exocyst, a highly conserved eight protein trafficking complex was identified in cultured adipocytes as a key regulator of GLUT4 trafficking in response to insulin. However, it is not known if the molecular mechanisms through which the exocyst orchestrates exocytosis of this glucose transporter in adipocytes are conserved in other, insulin-responsive tissues, such as the skeletal muscle. Skeletal muscle cells are responsible for the vast majority of insulin-induced glucose uptake, therefore investigating glucose transporter trafficking in these cells will be key to better understanding the mechanism of glucose homeostasis. Sec10 is a central subunit of the exocyst complex, and its overexpression can lead to overall increased exocyst activity, while its knockdown leads to protein degradation of several of the other subunits. We have recently generated a new transgenic mouse to analyze exocyst-regulated cellular trafficking in vivo: the first mouse strain with a conditional Sec10 allele. This unique model enables us to investigate the consequences of tissue-specific Sec10 inactivation by facilitating generation of adipose and muscle-specific conditional Sec10 knockout animals. We hypothesize that in vivo modulation of the exocyst complex can independently regulate GLUT4 exocytosis and glucose uptake in insulin-responsive cells and tissues, affecting the diabetic phenotype. To test this hypothesis, we propose the following Specific Aims: (1) Determine if the exocyst mediated regulatory mechanism of insulin-induced GLUT4 exocytosis is conserved in skeletal muscle cells. (2) Test for defects in glucose homeostasis in vivo using adipocyte and skeletal muscle-specific Sec10 knockout mouse strains. (3) Determine T2DM-associated metabolic defects in mice can be ameliorated by increasing GLUT4 exocytosis with a novel tissue-specific in vivo gene delivery system. The anticipated outcome of this project is the first in vivo evidence of the exocyst's role in regulating insulin-induced glucose uptake, and verification whether this regulatory mechanism is conserved in muscle tissues as well. These studies will also show if the exocyst, and other molecules that directly control GLUT4 exocytosis, are potential therapeutic targets for insulin resistance and diabetes.